CN101402674A - Functional peptide segment of epididymis protease inhibitors and uses thereof - Google Patents
Functional peptide segment of epididymis protease inhibitors and uses thereof Download PDFInfo
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Abstract
The invention discloses an epididymis protease inhibitor which is a functional peptide segment of Eppin. The inhibitor is any one selected from amino acid sequences shown from SEQ ID No.1 to SEQ ID No.7 and has advantages of single component, simple structure and strong pertinence of immunoreaction inspired; polypeptide with at least 80 percent of sequences the same as the functional peptide segment of Eppin can be used for constructing immunogen which can improve the immunogenicity of the functional peptide segment of Eppin, stimulate an organism to generate a specific antibody, reduce Eppin level in blood plasma and induce high-efficient, safe and reversible antifertility effect. The immunogen of the epididymis protease inhibitor can form a medicament composite with medically acceptable carriers, and the composite can be used for preparing male contraception vaccines, therefore, the epididymis protease inhibitor has broad application prospect.
Description
Technical field
The present invention relates to biological technical field, particularly the functional peptide fragment of epididymis protease inhibitors and application.
Background technology
Population, resource and environmental problem are to have a strong impact on human survival and the key issue of socio-economic development in this century, and the research that avoids conception and control birth is subjected to very big attention.Up to now, the male contraception method is confined to also that traditional external row is smart, sterilization and use condom etc., does not still have the new measure appearance of practising contraception of efficient, convenience, safety, reversible.
In decades, it is target antigen that the investigator attempts with reproduction associated hormone and sperm-specific protein, immunization method is applied to male fertility control, but still there are problems, as not finding best specific target antigen as yet, the poor antigen of natural antigen and immunological tolerance, the immunological contraception effect is undesirable etc.The huge obstacle that these problems have become the pregnancy vaccine development and used.
Calendar year 2001, (epididymal protease inhibitor Eppin) is found the human epididymal proteinase inhibitor first, and it is predominant expression in testis and epididymis, mainly is distributed in sperm surface and the refining.Reversible sterile (contraceptive prevalence rate 7/9 appears in the male rhesus macaque with the Eppin truncated protein immunity that keeps carboxyl terminal, reversible rate 5/7), semen analysis finds that the propulsion ability of sperm weakens, and the male sex hormone level is unaffected in the quantity of sperm and the body, also do not see the generation of autoimmune disorder, show that Eppin is a kind of extremely rising target antigen, but it is an autoantigen, so still there are problems such as the weak and immunological tolerance of immunogenicity, in addition, the bad peptide section that may cause toxic side effects at a specified future date among the Eppin is not done rejecting or processing.
Summary of the invention
In view of this, one of purpose of the present invention is to provide the functional peptide fragment of Eppin, is selected from any in the aminoacid sequence shown in SEQ ID No.1 to the SEQ ID No.7.
Two of purpose of the present invention is to provide a kind of immunogen, comprises the polypeptide that has at least 80% sequence homogeny with the functional peptide fragment of described Eppin.
Further, the functional peptide fragment that comprises described Eppin;
Further, functional peptide fragment and the carrier protein couplet by Eppin forms;
Further, described carrier proteins is selected from keyhole limpet hemocyanin or bovine serum albumin.
Three of purpose of the present invention is to provide a kind of pharmaceutical composition, comprises the described immunogen and the pharmaceutically acceptable carrier of immunology significant quantity.
Four of purpose of the present invention be to provide can with the functional peptide fragment specificity bonded antibody of described Eppin.
Five of purpose of the present invention is to provide the application of functional peptide fragment in the preparation immunogen of described Eppin.
Six of purpose of the present invention is to provide the application of described pharmaceutical composition in preparation male contraception vaccine.
Beneficial effect of the present invention is: the invention discloses the functional peptide fragment of Eppin, be selected from any in the aminoacid sequence shown in SEQ ID No.1 to the SEQ ID No.7, the immune response single, simple in structure, that excite of its composition is with strong points; The polypeptide that has at least 80% sequence homogeny with the functional peptide fragment of described Eppin can be used for making up immunogen, it can improve the immunogenicity of the functional peptide fragment of Eppin, stimulate body to produce specific antibody, reduce Eppin level in the serum, inducement efficient, safety, reversible antifertility action; Immunogen of the present invention can become pharmaceutical composition with pharmaceutically acceptable vehicle group, be used to prepare the male contraception vaccine, for the clinical immunotherapy of male contraception provides new means, have important theory and be worth and wide application prospect, can produce important social benefit and economic benefit.
Description of drawings
In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention is described in further detail below in conjunction with accompanying drawing, wherein:
1.5% agarose gel electrophoresis figure of the positive cloned plasmids pPET-32a-Eppin of Fig. 1 double digestion product;
Fig. 2 is the 12%SDS-PAGE figure of Eppin engineering bacterium expression product;
Fig. 3 is the 12%SDS-PAGE figure of reorganization Eppin;
Fig. 4 is the 12%SDS-PAGE figure of anti-Eppin polyclonal antibody.
Embodiment
The present invention adopts dislocation to repeat the shell type method, Eppin is divided into 32 polypeptide fragments, again each polypeptide fragment and carrier protein couplet are made 32 immunogens, the external anti-Eppin specific antibody identification of each immune original work detects, again each immunogen is formed composition with Freund's complete adjuvant or Freund's incomplete adjuvant respectively, with this composition immunity male mice, carry out immunogenicity detection and antifertility capability study in the body, it is strong to filter out several immunogenicities thus, antibody produces stable, immunogen with efficient antifertility ability, thereby obtain the functional peptide fragment of Eppin, be selected from any in the aminoacid sequence shown in SEQ ID No.1 to the SEQ ID No.7, its composition is single, simple in structure, the immune response that excites is with strong points.
The present invention includes the polypeptide that has at least 80% sequence homogeny with the functional peptide fragment of described Eppin.In the art, the polypeptide with at least 80% sequence homogeny can keep the biologic activity of former peptide section basically.These polypeptide include but not limited to: the polypeptide that (1) has one or several amino-acid residue to be substituted, to lack or/and insert, and replacement or the amino-acid residue that inserts can be also can not encoded by genetic code; (2) in one or more amino-acid residues, has the polypeptide of substituted radical.According to the instruction of this paper, these polypeptide belong to the scope of well known to a person skilled in the art.
The present invention includes a kind of immunogen, it comprises the polypeptide that has at least 80% sequence homogeny with the functional peptide fragment of Eppin.The immunogenic immunology significant quantity of the present invention can be according to variations such as route of administration, patient's age, body weight, healthy state and individual reaction, and dosage is every kg body weight 1-5 milligram usually, and this dosage can be used by one or many.
The present invention also comprises a kind of pharmaceutical composition, and described immunogen and pharmaceutically acceptable carrier that it comprises the immunology significant quantity can be used for preparing medicament forms such as male contraception vaccine.Described pharmaceutically acceptable carrier generally should be compatible with immunogen and can not be harmful to acceptor, for example Freund's complete adjuvant, Freund's incomplete adjuvant etc.Those of ordinary skills can determine to comprise quick-release or/and sustained release preparation by pharmaceutical dosage form at an easy rate.Various formulations can be according to the conventional production method preparation of pharmaceutical field.The gained pharmaceutical preparation can be used by any approach easily, comprise subcutaneous, oral, intramuscular, endoperitoneal, or other approach parenteral or enteron aisle.
Hereinafter with reference to accompanying drawing, the preferred embodiments of the present invention are described in detail.
One, the screening of the functional peptide fragment of Eppin and evaluation
1, the preparation of reorganization Eppin
(1) structure of Eppin recombinant expression vector
According to Eppin conserved regions design and synthetic following primer: F1:5 '-atgggatcttctggacttttgag-3 ', R1:5 '-tcagggaaagcgtttattcttgc-3 '; With healthy people's testis tissue cDNA (Clontech company) is template, with F1 and R1 is that the upstream and downstream primer carries out pcr amplification, the PCR product is to cut glue after 1.5% agarose gel electrophoresis is identified to reclaim purifying through mass percentage concentration, the PCR product of purifying is inserted carrier pCR 2.1-TOPO TA (Invitrigon company), be transformed into CaCl again
2The DH5 α competent escherichia coli cell of method preparation is used the LB plate screening positive colony that contains penbritin, extracts plasmid, and enzyme cutting method is identified positive colony plasmid and order-checking, obtains long 402bp, comprises the regional Eppin gene of whole open reading frame (ORF);
According to Eppin gene ORF sequence, design and synthesize following primer: F2:5 '-cgc
GgatccGgatcttctggacttttgagcctc-3 ', underscore partly are BamH I restriction enzyme site; R2:5 '-ccc
AagcttGggaaagcgtttattcttgcaggtg-3 ', underscore partly are Hind III restriction enzyme site; With the positive colony plasmid that obtains previously is template, with F2 and R2 is last, downstream primer carries out pcr amplification, the PCR product is through identifying, use BamH I and Hind III double digestion behind the purifying, be connected through the prokaryotic expression carrier PET-32a of BamH I and HindIII double digestion again with equally, connect product and be transformed into DH5 α competent escherichia coli cell, with the LB plate screening positive colony that contains penbritin, extract plasmid, enzyme cutting method is identified the positive colony plasmid, the agarose gel electrophoresis figure of double digestion product is as shown in Figure 1: 1 swimming lane is λ-dna molecular amount standard (λ-EcoT14 I digest DNA Marker that EcoT14 I enzyme is cut, TAKARA company), 2 swimming lanes are dna molecular amount standard, 3 swimming lanes are the double digestion product, the double digestion product shows two DNA bands as a result, wherein a treaty 400bp is consistent with the purpose clip size; To identify correct positive colony plasmid order-checking, the positive colony plasmid that sequence order is correct is the recombinant expression vector pPET-32a-Eppin that successfully makes up;
(2) structure of Eppin engineering bacteria
PPET-32a-Eppin is transformed into BL21 (DE3) competent escherichia coli cell with step (1) gained recombinant expression vector, with the LB plate screening positive colony that contains penbritin, extract plasmid, after the enzyme cutting method preliminary evaluation has or not the insertion fragment, guarantee that by sequencing direction of insertion is correct again, the correct positive colony of plasmid sequence is the Eppin engineering bacteria that successfully makes up;
(3) abduction delivering of Eppin
Step (2) gained Eppin engineering bacteria is inoculated in 100mL to be contained in the liquid LB substratum of penbritin that concentration is 50 μ g/mL, in 37 ℃ of temperature, 250r/min jolting overnight incubation, gained bacterium liquid is changed over to by 1: 100 (volume ratio) in the liquid LB substratum that is preheated to 37 ℃ of temperature, continue jolting and be cultured to A
600Reach 0.6, adding final concentration is sec.-propyl-β-D thiogalactoside (IPTG) inductor of 0.01mol/L, continue to cultivate after 3 hours, in 4 ℃ of temperature, centrifugal 10 minutes of 5000g, collect bacterial precipitation, adding 1 * binding buffer liquid in ice bath (is that concentration is 20mmol/L, the pH value is 7.9 Tris-HCl damping fluid, contain the N,O-Diacetylmuramidase that concentration is 100 μ g/mL, concentration is the urea of 6mol/L, concentration is that imidazoles and the concentration of 5mmol/L is the NaCl of 0.5mol/L), mixing, ultrasonication bacterium in short-term in ice bath, again in 4 ℃ of temperature, 15, centrifugal 20 minutes of 000g collects supernatant liquor;
Get supernatant liquor, with mass percentage concentration is that 12% separation gel carries out sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), SDS-PAGE schemes as shown in Figure 2: 1 swimming lane is a protein molecular weight standard, 2 swimming lanes are the cracking supernatant of the Eppin engineering bacteria after IPTG induces, 3 swimming lanes are the colibacillary cracking supernatant of BL21 (DE3) before IPTG induces, from figure as can be known, the Eppin engineering bacteria efficiently expresses Eppin under IPTG induces, expression product is the fusion rotein of relative molecular mass 20000, comprises 6 Histidines (His) label that recombinant expression vector is introduced at the Eppin carboxyl terminal.
(4) purifying of Eppin
Get the supernatant liquor that step (3) is collected, 6 His labels that utilize recombinant expression vector to introduce at the Eppin carboxyl terminal are with Ni
2+-NTA affinity column (Novagen company) carries out purifying, according to the described step operation of chromatography column specification sheets, (be that concentration is 20mmol/L with elutriant A earlier, the pH value is 7.9 Tris-HCl damping fluid, contain the N,O-Diacetylmuramidase that concentration is 100 μ g/mL, concentration is the urea of 8mol/L, concentration is that imidazoles and the concentration of 400mmol/L is the NaCl of 0.5mol/L) wash-out non-specific binding albumen, (be that concentration is 20mmol/L with elutriant B again, the pH value is 7.9 Tris-HCl damping fluid, containing imidazoles and the concentration that concentration is 1mol/L is the NaCl of 0.5mol/L) the wash-out target protein, be 0.1mol/L at last with concentration, the pH value is that 7.5 phosphate buffered saline buffer (PBS) is dialysed to the target protein that wash-out goes out, make protein renaturation, promptly get the Eppin that recombinates;
Getting reorganization Eppin, is that 12% separation gel carries out SDS-PAGE with mass percentage concentration, and SDS-PAGE scheme as shown in Figure 3: 1 swimming lane is the Eppin that recombinates, and 2 swimming lanes are protein molecular weight standard; From figure as can be known, higher through the reorganization Eppin of aforesaid method purifying purity of protein.
2, the preparation of anti-Eppin specific antibody
(1) anti-Eppin Polyclonal Antibody Preparation
With concentration is that the reorganization Eppin of 60 μ g/mL mixes with Freund's complete adjuvant or Freund's incomplete adjuvant equal-volume respectively, fully emulsified, makes immunogen, 2 of the healthy male new zealand rabbits of immune body weight 2.5-3kg.The immunity step be: first immunisation, with Eppin-Freund's complete adjuvant immunogen in the subcutaneous multi-point injection of rabbit back, every 30 μ g (1mL); 1 week at interval, booster immunization, with Eppin-Freund's incomplete adjuvant immunogen in the subcutaneous multi-point injection of rabbit back, every 30 μ g (1mL); Again 2 weeks of interval, repeat booster immunization 1 time; 1 week after the last immunity, detect antigen with reorganization Eppin, measure the immunize rabbit serum antibody titer with indirect enzyme-linked immunosorbent absorption (ELISA) method, the antagonist titre reaches the rabbit row carotid artery blood sampling more than ten thousand in 1: 51.2, separation of serum, (Pierce company) carries out purifying with the albumin A affinity column, promptly gets to resist the Eppin polyclonal antibody;
Get anti-Eppin polyclonal antibody, with mass percentage concentration is that 12% separation gel carries out SDS-PAGE, SDS-PAGE schemes as shown in Figure 4: 1 and 4 swimming lanes are respectively protein molecular weight standard, 2 and 3 swimming lanes are respectively the anti-Eppin polyclonal antibody of two strain purifying, from figure as can be known, through the anti-Eppin polyclonal antibody purity height of aforesaid method purifying.
(2) anti-Eppin MONOCLONAL ANTIBODIES SPECIFIC FOR
With concentration is that the reorganization Eppin of 60 μ g/mL mixes with Freund's complete adjuvant or Freund's incomplete adjuvant equal-volume respectively, fully emulsified, makes immunogen, the male Balb/c mouse in cleaning level 4-6 age in week of immune body weight 18-20g; The immunity step is: first immunisation,, abdominal cavity multi-point injection subcutaneous in mouse with Eppin-Freund's complete adjuvant immunogen, every 30 μ g (1mL); 2 weeks of interval, booster immunization,, abdominal cavity multi-point injection subcutaneous in mouse, every 30 μ g (1mL) with Eppin-Freund's incomplete adjuvant immunogen; Again 2 weeks of interval, repeat booster immunization 1 time; 2 weeks after the last immunity serve as to detect antigen with reorganization Eppin, measure the immune serum antibody titers with indirect elisa method, and the antagonist titre reaches 1: 12800 above mouse, not add the reorganization Eppin abdominal injection of adjuvant, every 30 μ g (1mL); Get mouse boosting cell after 3 days and SP2/0 myeloma cell is merged; Adopt irrelevant unlabelled antigen ELISA method to measure the titre of fused cell secretory antibody, to screen positive fused cell, the ELISA method is to detect antigen with reorganization Eppin, with irrelevant Protein G ST-Bax (i.e. the fusion rotein of being made up of Thiadiazolidine isomerase GST and apoptosis regulatory protein Bax) is blank, lure the negative contrast of label protein with the His sky, treat that the absorbance A value of gaging hole is judged to the positive more than or equal to 2.1 times of persons of negative control; The positive fused cell that filters out carries out next round again and cultivates and screen, and so forth, antibody-secreting positive rate until fused cell reaches more than 95%, obtain hybridoma cell strain 10 strains of the anti-Eppin monoclonal antibody of stably excreting altogether, induce ascites with the inoculation of gained hybridoma cell strain abdominal cavity through the pretreated male Balb/c mouse of pristane, gather ascites after 10 days, centrifugal, collect supernatant, remove by filter impurity, promptly get the ascites that contains anti-Eppin monoclonal antibody, the ascites that gained contains anti-Eppin monoclonal antibody adopts G albumen affinity column to carry out purifying, promptly gets to resist the Eppin monoclonal antibody.
3, the Eppin polypeptide fragment is synthetic
Aminoacid sequence according to Eppin, adopt dislocation to repeat the shell type method, hold carboxyl (C-) end to be decomposed into 32 polypeptide fragments from amino (N-) Eppin, the abbreviation and the sequence of each bar polypeptide fragment see table 1 for details, every polypeptide fragment comprises 10 amino-acid residues (fragment of the most close C end is 9 amino-acid residues), wherein 4 amino-acid residues are window, and 6 amino-acid residues are tumor-necrosis factor glycoproteins; Employing standard Fmoc scheme goes up synthetic above-mentioned 32 polypeptide fragments at solid-phase polypeptide synthesizer (ABI 431A type), identifies that through high performance liquid chromatography (HPLC) and mass spectroscopy (MS) purity of each polypeptide fragment of synthetic is all greater than 90%.
The abbreviation of table 1, Eppin polypeptide fragment and sequence
4, immunogenic preparation
In polypeptide fragment: the ratio of carrier proteins=40: 1 (mol ratio), 32 Eppin polypeptide fragments of synthetic are dissolved in 1mL water respectively, add 1-ethyl-3-(3-dimethylamino-propyl) phosphinylidyne diimmonium salt hydrochlorate (EDC) 10mg again, regulator solution pH value to 5, under room temperature, stirred 10 minutes, slowly add carrier proteins: keyhole limpet hemocyanin (KLH) 5mg, continue under the room temperature and stirred 3 hours, gained solution is 0.1mol/L with concentration, the pH value is that 7.5 PBS is in 4 ℃ of dialysed overnight of temperature, adjust coupling protein concentration to 1mg/mL, vacuum lyophilization promptly gets 32 immunogens.
It is carrier proteins that the present invention also can adopt bovine serum albumin (BSA), with reference to aforesaid method, with Eppin polypeptide fragment and BSA coupling, can make immunogen equally.
5, immunogenic screening and evaluation
(1) in-vitro screening and evaluation
Method: adopt indirect elisa method, 32 immunogens that make are diluted with the diluted liquid of bag (being that concentration is that 0.2mol/L, pH value are yellow soda ash-sodium bicarbonate buffer liquid of 9.2-10.7) respectively, make the coating buffer that concentration is 25 μ g/mL; Each coating buffer is added to respectively in the 96 hole Sptting plates, every hole 100 μ l, after putting 4 ℃ of bags of temperature and being spent the night, discard coating buffer, (in 1000mL PBS, add 0.5mLTween-20 and 0.1g Thiomersalate with washings, regulate pH value to 7.4) wash plate 3 times, add again that to be diluted to concentration expressed in percentage by volume with PBS be 5% calf serum confining liquid, every hole 100 μ L, put 37 ℃ of sealings of temperature 40 minutes, discard confining liquid, with washings washing 3 times, add again by anti-Eppin polyclonal antibody of the rabbit of 10 times of serial dilutions (extent of dilution is 1: 1000) or mouse anti Eppin monoclonal antibody (extent of dilution is 1: 1000), every hole 100 μ l, put after 37 ℃ of temperature hatch 1 hour, discard antibody-solutions, wash plate 3 times with washings, the goat anti-rabbit igg or the goat anti-mouse igg (extent of dilution is 1: 5000, Promega company) that add horseradish peroxidase (HRP) mark again, every hole 100 μ l, put after 37 ℃ of temperature hatch 45 minutes, discard traget antibody solution, wash plate 3 times, add tetramethyl biphenyl diamines (TMB) colour developing liquid again with washings, put 37 ℃ of temperature and hatch termination reaction after 10 minutes, measure the absorbance A value at 450nm wavelength place; Simultaneously, establish positive controls (reorganization Eppin), negative control group (KLH) and blank group (yellow soda ash-sodium bicarbonate buffer liquid); The result treats that with the three multiple equal value representations of hole A value gaging hole A value is judged to the positive more than or equal to 2.1 times of persons of negative control.
The result: KLH-YK and KLH-WC can be pointed out YK and WC may be the functional peptide fragment of Eppin by the anti-Eppin monoclonal antibody identification of 2 strains again by anti-Eppin polyclonal antibody identification in 32 immunogens.
(2) screening and evaluation in the body
Method: a, laboratory animal grouping: the male Balb/c mouse in cleaning level 4-6 age in week that is 18-20g with 100 body weight is divided into 10 groups (seeing Table 2) at random: totally 8 experimental group such as KLH-YK experimental group etc., positive controls (Eppin substitutes immunogen with reorganization) and negative control group (substituting immunogen) with KLH, 10 every group;
B, immunization protocol: it is that 0.1mol/L, pH value are among 7.5 the PBS that immunogen KLH-YK etc. is diluted in concentration respectively, be mixed with concentration and be the immunogen solution of 30 μ g/mL, again each immunogen solution is mixed with Freund's complete adjuvant or Freund's incomplete adjuvant equal-volume respectively, fully emulsified, make composition; The immunity step is: first immunisation, with immunogen-Freund's complete adjuvant composition injection mouse bilateral hind leg foot pad, every side 0.3 μ g (20 μ L); At interval 1 week, booster immunization is with immunogen-Freund's complete adjuvant composition injection mouse bilateral hind leg foot pad, every side 0.3 μ g (20 μ L); Again 2 weeks of interval, repeat booster immunization 1 time;
C, immunogenicity detect: before and after the first immunisation, and every interval certain hour, the blood sampling of row mouse tail vein, separation of serum adopts indirect elisa method to measure the titre of specific antibody in the immune male mouse serum;
D, antifertility ability detect: the 2nd week after the last immunity, mate experiment by 1: 1 capable male and female mouse of female-male proportion, and observe female mouse conceptual quotient and farrowing situation.
The result: specific antibody generates situation and female mouse conceptual quotient and farrowing situation and sees Table 2 in the immune male mouse serum, data presentation: the 3rd week after the first immunisation, except that the KLH-FT experimental group, can detect specific antibody in the male mouse serum of all the other each experimental group, antibody titers raises gradually afterwards, and the 6th week reached maximum after first immunisation; It is identical with experimental group that antibody in the male mouse serum of positive controls generates situation; No specific antibody generates in the male mouse serum of negative control group; Mate in the experiment the male and female mouse, except that the KLH-FT experimental group, the fecundity of male mouse all is suppressed after other each experimental group immunity, shows as the female mouse conceptual quotient and the negative control group that mate with it and compares, and significantly descends.
Specific antibody generates situation and female mouse conceptual quotient and farrowing situation in table 2, the immune male mouse serum
Based on above-mentioned qualification result, can draw as drawing a conclusion: all can stimulate body to produce specific antibody in (1) 32 polypeptide fragment behind YK (SEQ IDNo.1), WC (SEQ ID No.2), KV (SEQ ID No.3), TC (SEQ ID No.4), FN (SEQ ID No.5), GQ (SEQ ID No.6) and NN (SEQ ID No.7) and the carrier protein couplet, can be used for preparing immunogen; KLH-YK, KLH-WC, KLH-KV, KLH-TC, KLH-FN, KLH-GQ and KLH-NN can not only stimulate body to produce specific antibody in (2) 32 immunogens, and can induce antifertility action efficiently, can be used for preparing the male contraception vaccine; 3) by comparing the antifertility effect of each polypeptide fragment, can find that the effect of TC and FN is more outstanding.
Explanation is at last, above embodiment is only unrestricted in order to technical scheme of the present invention to be described, although by invention has been described with reference to the preferred embodiments of the present invention, but those of ordinary skill in the art is to be understood that, can make various changes to it in the form and details, and the spirit and scope of the present invention that do not depart from appended claims and limited.
Sequence table
＜110〉Military Medical Univ No.3, P.L.A
＜120〉functional peptide fragment of epididymis protease inhibitors and application
<160>7
<210>1
<211>10
<212>PRT
＜213〉homo sapiens (Human)
<400>1
Tyr?Phe?Leu?His?Trp?Trp?Tyr?Asp?Lys?Lys
1 5 10
<210>2
<211>10
<212>PRT
＜213〉homo sapiens (Human)
<400>2
Trp?Trp?Tyr?Asp?Lys?Lys?Asp?Asn?Thr?Cys
1 5 10
<210>3
<211>10
<212>PRT
＜213〉homo sapiens (Human)
<400>3
Lys?Lys?Asp?Asn?Thr?Cys?Ser?Met?Phe?Val
1 5 10
<210>4
<211>10
<212>PRT
＜213〉homo sapiens (Human)
<400>4
Thr?Cys?Ser?Met?Phe?Val?Tyr?Gly?Gly?Cys
1 5 10
<210>5
<211>10
<212>PRT
＜213〉homo sapiens (Human)
<400>5
Phe?Val?Tyr?Gly?Gly?Cys?Gln?Gly?Asn?Asn
1 5 10
<210>6
<211>10
<212>PRT
＜213〉homo sapiens (Human)
<400>6
Gly?Cys?Gln?Gly?Asn?Asn?Asn?Asn?Phe?Gln
1 5 10
<210>7
<211>10
<212>PRT
＜213〉homo sapiens (Human)
<400>7
Asn?Asn?Asn?Asn?Phe?Gln?Ser?Lys?Ala?Asn
1 5 10
Claims (9)
1, the functional peptide fragment of epididymis protease inhibitors is selected from any in the aminoacid sequence shown in SEQ ID No.1 to the SEQ ID No.7.
2, immunogen is characterized in that: comprise the polypeptide that has at least 80% sequence homogeny with the functional peptide fragment of the described epididymis protease inhibitors of claim 1.
3, immunogen according to claim 2 is characterized in that: the functional peptide fragment that comprises the described epididymis protease inhibitors of claim 1.
4, immunogen according to claim 3 is characterized in that: functional peptide fragment and carrier protein couplet by epididymis protease inhibitors form.
5, immunogen according to claim 4 is characterized in that: described carrier proteins is selected from keyhole limpet hemocyanin or bovine serum albumin.
6, pharmaceutical composition is characterized in that: the described immunogen of claim 2 and the pharmaceutically acceptable carrier that comprise the immunology significant quantity.
7, can with the functional peptide fragment specificity bonded antibody of the described epididymis protease inhibitors of claim 1.
8, the application of the functional peptide fragment of the described epididymis protease inhibitors of claim 1 in the preparation immunogen.
9, the application of the described pharmaceutical composition of claim 6 in preparation male contraception vaccine.
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| CN103819563A (en) * | 2013-12-31 | 2014-05-28 | 中国人民解放军第三军医大学第一附属医院 | Fusion protein MBP-Eppin, protein Eppin and preparation methods thereof |
| WO2015138919A1 (en) * | 2014-03-14 | 2015-09-17 | The University Of North Carolina At Chapel Hill | Small molecules for inhibiting male fertility |
| CN107490680A (en) * | 2016-06-09 | 2017-12-19 | 常州博闻迪医药科技有限公司 | A kind of Blood glycated haemoglobin enzyme-linked immune detection method |
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| RU2007108538A (en) * | 2004-08-11 | 2008-09-20 | Трабьон Фармасьютикалз, Инк. (Us) | LINKED PROTEIN DOMAIN |
| MX2008011350A (en) * | 2006-03-08 | 2008-09-23 | Bayer Healthcare Ag | Chimeric kunitz domains and their use. |
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2008
- 2008-10-20 CN CN2008102328922A patent/CN101402674B/en not_active Expired - Fee Related
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| CN101816798B (en) * | 2010-04-09 | 2011-10-05 | 中国人民解放军第三军医大学 | Gene vaccine of sperm-specific protein Ropporin and preparation method thereof |
| CN103539859A (en) * | 2013-09-23 | 2014-01-29 | 中国人民解放军第三军医大学第一附属医院 | Self-assembled polypeptide nanometer for preparing contraception vaccine |
| CN103539859B (en) * | 2013-09-23 | 2016-06-08 | 中国人民解放军第三军医大学第一附属医院 | For the preparation of the self assembly polypeptide nano of pregnancy vaccine |
| CN103819563A (en) * | 2013-12-31 | 2014-05-28 | 中国人民解放军第三军医大学第一附属医院 | Fusion protein MBP-Eppin, protein Eppin and preparation methods thereof |
| WO2015138919A1 (en) * | 2014-03-14 | 2015-09-17 | The University Of North Carolina At Chapel Hill | Small molecules for inhibiting male fertility |
| CN106414415A (en) * | 2014-03-14 | 2017-02-15 | 北卡罗来纳大学教堂山分校 | Small molecules for inhibiting male fertility |
| US10472334B2 (en) | 2014-03-14 | 2019-11-12 | The University Of North Carolina At Chapel Hill | Small molecules for inhibiting male fertility |
| CN111620830A (en) * | 2014-03-14 | 2020-09-04 | 北卡罗来纳大学教堂山分校 | Small molecules for inhibiting male fertility |
| CN107490680A (en) * | 2016-06-09 | 2017-12-19 | 常州博闻迪医药科技有限公司 | A kind of Blood glycated haemoglobin enzyme-linked immune detection method |
| CN108152511A (en) * | 2017-12-20 | 2018-06-12 | 广州瑞辉生物科技股份有限公司 | Coxsack A16 virus antigen polypeptides and its IgM antibody detection kit |
| CN108169484A (en) * | 2017-12-20 | 2018-06-15 | 广州瑞辉生物科技股份有限公司 | EV71 virus antigen polypeptides and its IgM antibody detection kit |
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